Phenothiazines, phenazines and phenoxazines are selectively taken up by cancer cells in living, unfixed tissue, and have been used both for the demarcation of tumor cells within tissue and evaluated as agents for potentially treating cancer. One of the oldest of these compounds is methylene blue. Phenothiazine and thiothixene derivatives have been used as antipsychotic agents for over 60 years, and include the classical dopamine antagonists that preferentially bind to the family of dopamine receptors (DR1-5). These phenothiazine-based dopamine receptor antagonists are among several types of so-called first generation antipsychotics which include chlorpromazine, fluphenazine, haloperidol, loxapine, mesoridazine, molindone, perphenazine, pimozide, thioridazine, thiothixene, and trifluoperazine that work by antagonism of dopamine (D2) receptors throughout the brain. High levels of cardiotoxicity reported for phenothiazine-based dopamine receptor antagonists have unfortunately limited their clinical use in all but the most serious circumstances. Although phenothiazine-based dopamine receptor antagonists have considerable potential for their clinical utility outside of treating psychoses, the clinical use of these phenothiazine compounds is severely limited due to their dose-limiting toxicity. Thioridazine (Mellaril®), for example, was removed from the market because of its cardiotoxicity in patients, believed to be caused by the excessive prolongation of QT interval found, and a direct result of its high hERG liability.
Substituted phenothiazines such as those found in the first generation antipsychotics are tricyclic heteroaromatic lipophilic compounds having a planar aromatic ring structure with a cationic center disposed adjacent the ring nitrogen that provides the basis for dopamine receptor and cytochrome P450 binding. This particular chemical structure has proven to be valuable in treating tumors, which has been demonstrated for decades in numerous scientific reports. Phenothiazine compounds such as chlorpromazine, fluphenazine, thioridazine and promazine inhibit tumor growth in a number of cell systems both in vitro and in vivo. Various phenothiazine compounds have demonstrated selective and preferential uptake by the P-glycoprotein transporter (P-gp) that is overexpressed in tumor cells. Various phenothiazine compounds have also demonstrated inhibition of multiple protein kinases in the PI3K/Akt/mTOR pathway, restoration and enhancement of cytotoxicity toward drug-resistant tumors, inhibition of the rapid proliferation of cycling cells through control of DNA replication, mitotic arrest and accumulation of monopolar spindles (e.g. KSP/Eg5), activation of caspase-3, and activity against MRSA and intracellular methicillin-susceptible S. aureus (MSSA). It has also been reported that multidrug resistance can be modulated by phenothiazine compounds, and that phenothiazine compounds specifically modulate P-gp mediated drug transport (Wang et al. Basic Clinical Pharmacology and Toxicology 103(4): 336-341 (2008); Liu et al. Journal of the National Cancer Institute 89(20): 1524-1529 (1997); Tuyander et al. PNAS 101(43):15364-15369 (2004); Lee et al. Cancer Research 67 (23):11359-11367 (2007)).
A major problem in treating many cancers is tumor heterogeneity that prevents a complete cytotoxic response of cancer cells to any particular treatment; whether this resistance to therapy is an intrinsic characteristic of the cancer cell type or is acquired through genetic mutation, drug therapy or epithelial-to-mesenchymal transitions (metastases). Examples of intrinsically resistant tumors that have a genetic mutation include the BRAFV600E tumors found in metastatic melanoma cells. Drug-induced resistance can occur with conventional chemotherapy drugs such as doxorubicin and tamoxifen used in the treatment of breast cancer, and a number of drugs in other cancers. Metastatic tumor cells that have been transformed to mesenchymal-like tumor cells are generally resistant to conventional chemotherapy, are highly aggressive, and have unique biological and morphological characteristic that differ substantially from tumor cells in the primary tumor. While successful breast cancer treatments can control estrogen-positive tumor cells in primary tumors, they are unable to control estrogen-negative metastatic tumor cells. Finally, cancer initiating cells that are highly resistant to conventional therapies are often found in breast cancer as discrete populations of mammary cells have been isolated on the basis of cell-surface markers and a subpopulation of Lin-CD44+CD24−/Low cells. (Al-Hajj M, Becker M W, Wicha M, Weissman I and Clarket M F, Curr. Opin. Genet. Dev. 2004 February; 14(1):43-47; Sheridan et al. Breast Cancer Research 8: R59 (2006; Isom et al. Human Pathology 43(3): 364-373 (2012); Kawaguchi et al. Breast Cancer Symposium Abstract No. 40 American Society of Clinical Oncology (2010)).
Overcoming the resistance acquired by specific types of tumor cells is difficult because they generally have a high content of P-gp on their cell surface membrane. Phenothiazine-based compounds have demonstrated efficacy in treating epithelial, mesenchymal-type metastatic, solid and hematopoietic tumors containing stem cell-like (CD44+/ALDH+; CD133/ALDH+) populations as well drug-resistant tumor cells containing elevated levels of P-gp. Thioridazine has demonstrated the ability to inhibit the growth of tumor cells and inducing apoptosis without affecting the growth of normal cells (Byun H J et al. Microvascular Research 84: 227-234 (2012); Gil-Ad I et al. Oncology Reports 15: 107-112 (2006); Sachlos et al. Cell 149:1-14 (2012)).
The phosphatidylinositol 3-kinase (PI3K/Akt/mTOR) signaling pathway is a key regulator of physiological cell processes which include proliferation, differentiation, apoptosis, motility, metabolism, and autophagy. Aberrantly upregulated PI3K/Akt/mTOR signaling characterizes many types of cancers where it negatively influences prognosis. Cancer stem cells are more sensitive to PI3K/Akt/mTOR pathway inhibition in hematological and solid tumors with small molecules when compared to healthy stem cells (Georgescu et al. Genes & Cancer 1(12):1170-1177 (2011); Prochownik US 20100298352). Thioridazine and related phenothiazines have been reported to successfully inhibited phosphorylation of kinases upstream and downstream of Akt, including phosphorylation of PDk1, FOXO, Akt, mTOR1, mTOR2, 4E-BP1 and p70S6K. These reports suggest that thioridazine effectively suppresses tumor growth activity by targeting the PI3K/Akt/mTOR/p70S6K signaling pathway; however, phenothiazines are highly selective and do not inhibit the activation of EGFR, or extracellular signal-regulated kinase 1/2 (ERK1/2) (Choi et al Annals of the New York Academy of Sciences 1138: 393-403 (2008); William Sellers US Army Medical Research Grant W81XWH-04-1-0169 (2007 Report); Dhawan et al. Molecular Cancer Therapeutics 10(11) Supplement 1 Abstract A218 (2011); Kang et al. Apoptosis Mar. 30, 2012); Kau et al. Cancer Cell 4:463-476 (December 2003)).
Using suppression of pluripotency transcription factors such as Octamer 4 (Oct4), the phenothiazine compound thioridazine was reported to selectively target dopamine receptors on cancer somatic stem cells that are involved with the initiation of leukemic disease and on breast cancer cells. Treatment of these cancer somatic stem cells with thioridazine demonstrated cytotoxicity toward cancer stem cells while demonstrating no cytotoxicity to normal human pluripotent stem cells. (Sachlos et al. Cell 149:1-14 (2012)). The authors of this study speculated that because neoplastic pluripotent stem cells express dopamine receptors (D1-D5) and human pluripotent stem cells do not, this drug could selectively target cancer stem cells.
There is clearly a need for safer drugs that can be taken up by cancer cells, modulate the multidrug transporter system, inhibit the activated PI3K/Akt/mTOR/p70S6K signaling pathway, control cancer stem cells and safely control tumor growth, proliferation, differentiation, apoptosis, motility, or autophagy in patients.
It is an object of the present invention therefor to provide compounds useful for treating cancer patients that advantageously control therapy-resistant tumor cells and have low cardiotoxicity risk.
It is a further object of the present invention to provide compounds that modulate the P-glycoprotein transporter system, inhibit the activated AKT signaling pathway, and control tumor growth, proliferation, differentiation, apoptosis, motility, or autophagy in patients.
It is a further object of the present invention to modulate the P-glycoprotein transporter system, inhibit the activated AKT signaling pathway, and control tumor growth, proliferation, differentiation, apoptosis, motility, or autophagy in patients with cancer with noncardiotoxic phenothiazine compounds.
It is also an object of the present invention to provide a method for treating a subject in need of cancer therapy comprising administering to the subject a compound in an amount effective in inhibiting tumor growth, proliferation, differentiation, apoptosis, motility, or autophagy in patients.